Transgenic animals

Abstract

The present invention provides improved methods and compositions for the generation of transgenic non-human animals. The present invention permits the introduction of exogenous nucleic acid sequences into the genome of unfertilized eggs (e.g., pre-maturation oocytes and pre-fertilization oocytes) by microinjection of infectious retrovirus into the perivitelline space of the egg. The methods of the present invention provide an increased efficiency of production of transgenic animals with a reduced rate of generating animals which are mosaic for the presence of the transgene.

What is claimed is:  
     
       1. A method for expressing a protein of interest in a non-human mammal wherein said protein of interest is encoded by a polynucleotide contained within the genome of a recombinant retrovirus, and said polynucleotide is integrated into the genome of a non-human mammalian unfertilized oocyte, comprising the steps of 
       a) providing:  
       i) an unfertilized non-human mammalian egg comprising an oocyte having a plasma membrane and a zona pellucida, said plasma membrane and said zona pellucida defining a perivitelline space;  
       ii) an aqueous solution containing said recombinant retrovirus, wherein said recombinant retrovirus comprises said polynucleotide encoding said protein of interest;  
       b) introducing said solution containing said recombinant retrovirus into said perivitelline space under conditions which permit the infection of said oocyte to provide an infected oocyte;  
       c) contacting said infected oocyte with sperm under conditions which permit the fertilization of said infected oocyte to produce an embryo;  
       d) transferring said embryo into a hormonally synchronized non-human mammalian recipient animal;  
       e) allowing said embryo to develop into at least one viable transgenic mammalian animal, under conditions such that said protein of interest is expressed at detectable levels by said transgenic mammalian animal.  
     
     
       2. The method of claim  1 , wherein said unfertilized oocyte is a pre-maturation oocyte. 
     
     
       3. The method of claim  2 , further comprising following the introduction of said solution containing retrovirus into said pre-maturation oocyte, the further step of culturing said pre-maturation oocyte under conditions which permit the maturation of said pre-maturation oocyte. 
     
     
       4. The method of claim  1 , wherein said unfertilized oocyte is a pre-fertilization oocyte. 
     
     
       5. The method of claim  1 , further comprising identifying at least one transgenic non-human mammalian offspring. 
     
     
       6. The method of claim  1 , wherein said mammal is a bovine. 
     
     
       7. The method of claim  1 , wherein said recombinant retrovirus comprises Moloney murine leukemia virus long terminal repeat.